Production of genetically-controlled herbicide resistance in cotton plants in the absence of genetic engineering

ABSTRACT

A process is provided for selecting cotton plants which exhibit naturally-occurring genetically-controlled herbicide resistance. Initially cotton seeds are subjected to cold stress (as described) and cotton plants are formed from the seeds that germinate. Seeds are formed following the self-pollination of the resulting plants. These seeds upon maturity are soaked in a liquid containing a herbicide (e.g., a glyphosate) for a period of time sufficient for the herbicide to reach the embryos of the seeds. The seeds following such soaking are planted in a growing medium and produce at least one cotton plant that displays herbicide resistance. The plant is analyzed for the possible presence of a foreign gene for herbicide resistance that would attribute the manifest herbicide resistance to genetic engineering. A cotton plant is selected wherein the herbicide resistance is not attributable to genetic engineering.

BACKGROUND OF THE INVENTION

[0001] Cotton, (i.e., plants of the genus Gossypium) long has beenrecognized to be an important crop which is being grown in many parts ofthe world. This crop is primarily grown for its lint. The seed may beused for planting or as a source of an edible oil with the seed residueserving as a livestock feed.

[0002] Modern agricultural practices are increasingly taking advantageof herbicides to eliminate unwanted weeds from cotton fields and tominimize the labor expense of tilling the fields to eliminate weeds.Presently, no selective herbicides that will kill only the major weedspecies are available for use in cotton fields. Accordingly, it has beennecessary in the past to use genetic engineering to genetically modifythe cotton plants so that they are resistant to herbicides that arenormally non-selective and are effective in controlling the weeds thatappear in the growing area. Such herbicide can be applied by spraying tothe entire growing area at an appropriate time in the plant's lifecycle. Representative weeds that are killed by herbicides in cottonfields include Arnaranthis (Pigweed), Russian Thistle, Kochia, MintWeed, Field Bindweed, Silver Leaf Nightshade, Lambs Quarters, BurrRagweed, etc.

[0003] Genetic engineering has involved the incorporation of a foreigngene for herbicide resistance that is not naturally-occurring in cottoninto a chromosome of the cotton plant. Such procedure requires specialexpertise and tends to be costly. It is necessary to use a promoter thatis not naturally-occurring in cotton to be inserted so as to enable theforeign gene for herbicide resistance to be activated in the chromosomesof the cotton plant. A common promoter when incorporating herbicideresistance for glyphosate resistance into cotton is CaMV35S. Otheravailable promoters include ACTIN, NOS, and PCSLV. Representative priorpublications that concern the use of genetic engineering to produce suchherbicide resistance include U.S. Pat. Nos. 4,971,908; 5,145,783;5,312,910; 5,352,605; 5,530,196; 5,633,435; and 5,858,742.

[0004] It is an object of the present invention to provide a new routefor providing genetically-controlled herbicide resistance in cottonplants in the absence of genetic engineering involving the insertion ofa foreign gene in cotton plants.

[0005] It is an object of the present invention to provide a cotton seedcapable of forming a cotton plant having genetically-controlledherbicide resistance that is not attributable to genetic engineeringinvolving the insertion of a foreign gene in cotton plants.

[0006] It is an object of the present invention to provide a cottonplant having genetically-controlled herbicide resistance that is notattributable to genetic engineering involving the insertion of a foreigngene in cotton plants.

[0007] It is another object of the present invention to provide a newisolated nucleic acid encoding for a protein which when expressed causesherbicide resistance that is naturally-occurring in cotton.

[0008] It is another object of the present invention to provide anisolated nucleic acid comprising HG^(g) gene selected fromR418ctHG^(g)hg^(g) having ATCC Accession No PTA-2132 which whenexpressed causes a cotton plant to be glyphosate herbicide resistant, aswell as a to provide a vector and plant cell comprising the same.

[0009] It is a further object of the present invention to provide acotton plant having genetically-controlled herbicide resistance that canbe sprayed with a herbicide during all phases of the life cycle of theplant without any substantial harm.

[0010] These and other objects, as well as the scope, nature andutilization of the claimed invention will be apparent to those skilledin this area of technology from the following detailed description andappended claims.

SUMMARY OF THE INVENTION

[0011] A process is provided for selecting a cotton plant which exhibitsgenetically-controlled herbicide resistance that is not attributable togenetic engineering comprising:

[0012] (a) cold stressing cotton seeds in a humid atmosphere,

[0013] (b) planting the cotton seeds following step (a) to producecotton plants,

[0014] (c) self-pollinating cotton plants produced in step (b) andforming cotton seeds thereon as the result of the self-pollination,

[0015] (d) maintaining the cotton seeds produced in step (c) forsufficient time to reach maturity,

[0016] (e) soaking the mature cotton seeds from step (d) in a liquidcomprising a herbicide for a period of time sufficient for the herbicideto reach the embryos of the cotton seeds,

[0017] (f) planting the cotton seeds following the soaking of step (e)in a growing medium and producing at least one cotton plant thatdisplays resistance to the herbicide,

[0018] (g) analyzing a portion of a plant from at least one cotton plantproduced in step (f) or a descendant thereof to confirm the absence of aforeign gene for herbicide resistance introduced by genetic engineering,and

[0019] (h) selecting a cotton plant from step (g) which exhibitsgenetically-controlled herbicide resistance that is not attributable toa foreign gene for herbicide resistance introduced by geneticengineering.

[0020] A cotton seed is provided that is capable of forming a cottonplant having genetically-controlled glyphosate herbicide resistance thatis attributable to the homozygous gene pair HG^(g) HG^(g) obtainablefrom cotton R418ctHG^(g)hg^(g) having ATCC Accession No. PTA-2132.

[0021] A cotton plant is provided having genetically-controlledglyphosate resistance that is attributable to the homozygous gene pairHG^(g) HG^(g) obtainable from cotton R418ctHG^(g)hg^(g) having ATCCAccession No. PTA-2132.

[0022] An isolated nucleic acid comprising a HG^(g) gene derived fromcotton R418ctHG^(g)hg^(g) having ATCC Accession No. PTA-2132 is madepossible which when expressed in a cotton plant causes the cotton plantto be glyphosate herbicide resistant The isolated nucleic acid can beincorporated in a vector and the vector can be incorporated in a plantcell.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0023] Normally cotton seeds are adversely influenced when subjected tocold temperatures below 55° F. for any appreciable period of time (e.g.,over eight hours). When such cold temperatures are encountered, it isobserved that the plants resulting from such seeds commonly exhibitretarded growth, spindly stems, root abnormalities, and failure to reachsexual maturity.

[0024] The cotton seeds that are used as the starting material in theprocess of the present invention are harvested from cotton plants whichhave not been previously rendered herbicide resistant by the use ofgenetic engineering through the insertion of a foreign gene forherbicide resistance. In preferred embodiments, the cotton seeds arederived from cotton plants of preexisting cotton varieties or lineswhich are recognized to display superior agronomic characteristics underconventional cotton growing conditions.

[0025] During the initial step of the process of the present invention,the cotton seeds are cold stressed in a humid atmosphere underconditions that normally would be deleterious to such seeds.Representative temperatures within the range of approximately 40 to 50°F. (most preferably approximately 42 to 49° F.) commonly are employedduring the cold stressing. If the temperature is above 50° F., many moremarginal plants commonly survive and commonly require an excessivenumber of cold-stressing cycles for removal from the population. If thetemperature is below 40° F., there commonly are few surviving plants.During the cold stressing step of the process, day temperatures ofapproximately 49° F. and night temperatures of approximately 42° F. or aconstant temperature of approximately 45° F. have been used toadvantage. The duration of the cold stressing step of the processpreferably is at least approximately 7 days, and most preferably atleast approximately 18 days. Cold stressing of 7 to 18 days has commonlybeen utilized when practicing the process of the present invention. Thedesired results are not commonly achieved if the duration of the coldstressing is less than seven days. Colder temperatures for shorterperiods of time have proven to be ineffective in research conducted todate. During the cold stressing it is essential that the seedsadditionally be subjected to a humid atmosphere. The relative humiditypreferably should be at least 90 percent, and most preferably at least99 percent. The cold stressing optionally can be conducted in aplurality of generations. In a preferred embodiment a plurality ofgenerations (e.g., three generations) of cold stressing are utilizedwith the progeny of the surviving plants being subsequently coldstressed in each successful generation. This results in a stablepopulation of cold tolerant cotton plants.

[0026] At the conclusion of the cold stressing step the seeds areplanted, and an effort is made to germinate these to produce cottonplants. It is observed that the cold stressing generally has had adetrimental influence on the ability of most of the seeds to producetypical cotton plants as evidenced by the functional death of theseedling or germination of the seedling combined with an inability togrow to form a normal plant. For instance, some abnormal plants willreach a height of only approximately 6 to 10 inches and will grow nofurther. Even grow lights will not benefit these plants. Other plantswill reach a height of 6 to 10 inches and remain at that height for 2 to3 weeks prior to resuming normal growth. Maturity will be greatlyprolonged because of late flowering and late boll set. A minority of thecold-stressed plants will grow normally and will flower and boll set thesame as non-cold-stressed plants. It is these plants that are furtherused in the process of the present invention. Commonly, onlyapproximately 0.1 to 45 percent of the cotton seeds will germinate toform cotton plants following the cold stressing. The variation in thepercentage of cotton plants that grow normally following cold stressingvaries with the cotton type that is selected as the starting material.Delta cotton types display only a rare individual plant. The resultswith West Texas (High Plains) varieties vary greatly. For instance, intests conducted to date, Paymaster HS-26 had only an approximately 2percent survival rate, HS-200 had an approximately 8 percent survivalrate, JH216 had an approximately 40 percent survival rate, and Tejas hada 42 percent survival rate.

[0027] Cotton plants resulting from the germination of the cold-stressedseeds are selected for further use in accordance with the process of thepresent invention which are observed to demonstrate normal phenotypesand growth characteristics. These plants arc subjected to controlledself-pollination and cotton seeds are formed upon the resulting plantsthat are allowed to mature.

[0028] The resulting cotton seeds are harvested and are stored for asufficient period of time to break the natural dormancy of the seeds.For instance, this can be accomplished by placing the cotton seeds in afreezer at a temperature of 5° F. or less for a minimum of 72 hours andpreferably for at least one week.

[0029] The mature cotton seeds next are soaked in a liquid comprising aherbicide that normally will kill cotton plants when applied at aconcentration at least sufficient to kill unwanted weeds that commonlyoccur in cotton fields. Representative herbicides include glyphosate,2,4-dichlorophenoxyacetic acid, glufosinate ammonium butanoic acid,3,5-dibromo-4-hydroxybenzonitrile, etc.

[0030] In a preferred embodiment the herbicide is a glyphosate. Suchherbicide is N-(phosphonomethyl)glycine of the chemical formula:

[0031] and is commercially available from Monsanto Corporation under theROUNDUP trademark and other companies under various trademarks. Thisherbicide is a non-selective, broad spectrum, post-emergence herbicidewhich is registered for use in more than fifty crops. This molecule isan acid, which dissociates in aqueous solution to form phytotoxicanions. Several anionic forms are known As used herein, the name“glyphosate” refers to the acid and its anions. Glyphosate inhibits theshikimic acid pathway which provides a precursor for the synthesis ofaromatic amino acids. Specifically, glyphosate curbs the conversion ofphosphoenolpyruvate and 3-phosphoshikimic acid to5-enolpyruvyl-3-phosphoshikimic acid by inhibiting the enzyme5-enolpyruvyl-3-phosphoshikimiate synthase.

[0032] The 2,4-dichlorophenoxyacetic acid herbicide commonly is known as“2,4-D” and is commercially available from a number of sources,including United Agri Products, Incorporated. Such herbicide is known topromote very rapid plant growth that is not sustainable.

[0033] The glufosinate ammonium butanoic acid herbicide is commerciallyavailable from the Aventis Corporation under the LIBERTY trademark.

[0034] The 3,5-dibromo-4-hydroxybenzonitrile herbicide commonly iscommercially available from the Rhone Poulenc Corporation under theBUCTRIL trademark.

[0035] Preferably the cotton seeds are soaked in an aqueous solution ofthe herbicide. Commonly the herbicide is present in the solution in aconcentration of approximately 2 to 6 percent by weight, and mostpreferably in a concentration of approximately 2.5 percent by weight.Commonly the cotton seeds are simply immersed or suspended in the liquidcomprising the herbicide. The soaking of cotton seeds is conducted for aperiod of time that is at least sufficient for the herbicide to reachthe embryos of the cotton seeds A soaking time of at least 6 hours ispreferred and most preferably a soaking time of at least 8 hours is usedRepresentative soaking times for the cotton seeds commonly areapproximately 6 to 12 hours. The liquid comprising the herbicide can besimply provided at room temperature when the cotton seeds are in contactwith the liquid and are undergoing such soaking.

[0036] Following soaking in the herbicide, the resulting seeds areplanted in a growing medium (e.g., soil) and germination of the seeds isattempted to produce cotton plants that display herbicide resistance.The herbicide resistance can be confirmed by spraying the resultingcotton plants with the same herbicide in a concentration typically usedto kill weeds growing in a cotton field.

[0037] Alternatively, such screening of the resulting cotton plants forherbicide resistance can include the inclusion of the herbicide in thegrowing medium where the resulting seeds are planted. Good results areobtained in a preferred embodiment when one gallon of a solutioncontaining the herbicide in a concentration of approximately 2 to 6percent by weight is added to each 4 gallons of soil. The presence ofthe herbicide in the soil helps to assure that an a typical seed havinga harder seed coat has not given a false indication of herbicideresistance by its survival up to the point of germination.

[0038] It has been found that a small proportion of the seeds followingthe cold-stressing, soaking in a liquid comprising a herbicide, andplanting in a growth medium, will germinate and yield cotton plants thatexhibit resistance to the herbicide. The percentage of the plants thatwill grow normally at this step in the process has been found to varyfrom variety to variety. Some varieties have produced no survivingplants in tests to date. Some varieties have produced up toapproximately 1 surviving plant per 1,000 seeds, others approximately 1surviving plant per 5,000 seeds, and others approximately 1 survivingplant per 25,000 seeds. The herbicide resistance of the resulting plantscan be further confirmed by another contact (e.g., spraying) with theherbicide. A simple field test kit for herbicide resistance is availablefrom AIT Company of Iroquois, S. Dak., as well as other sources.

[0039] A portion of the herbicide-resistant cotton plant producedfollowing such germination or a descendant thereof next is analyzed toconfirm that the manifest herbicide resistance is not the result ofgenetic engineering involving the insertion of a foreign gene that isnot naturally-occurring in cotton into the cotton plant by man. Thispreferably is done by checking for the presence of a promoter that wasintroduced by man when inserting a foreign gene construct for herbicideresistance. This analysis is used to confirm that the subject cottonplant is not a genetically modified organism and that the manifestherbicide resistance is attributable to a naturally-occurring geneticbasis other than that introduced by genetic engineering. Morespecifically, this analysis is used to confirm that the resultingherbicide-resistant cotton plant or plants were not derived in somemanner (e.g., by outcrossing) from a cotton plant that has beengenetically engineered for herbicide resistance. In accordance with theprocess of the present invention a cotton plant is next selected inwhich the herbicide resistance is under genetic control and in whichthere is no evidence of the use of genetic engineering to produce theherbicide resistance, such as the presence of a promoter for suchherbicide resistance. Any suitable technique can be utilized to confirmthe absence of the use of genetic engineering to produce the herbicideresistance. For instance, a DNA-polymerase chain reaction can beutilized. In a preferred embodiment a DNA-polymerase chain reaction iscarried out on a portion of a cotton plant leaf. This analysis can becarried out to advantage when analyzing a portion of a young growingleaf. A DNA sequence analysis can be utilized to confirm that the genefor herbicide resistance does not conform to the sequence of a foreigngene inserted into the cotton genome by genetic engineering. Forinstance, it can be confirmed that the foreign Petunia and CP4 genesheretofore incorporated into commercially available cotton varieties toimpart herbicide resistance are absent.

[0040] Also, contemplated by the instant invention are the nucleic acidswhich comprise the genes which when expressed in the cotton plantprovide herbicide resistance to that plant. Once a cotton plant whichexhibits genetically-controlled herbicide resistance that is notattributable to genetic engineering has been identified, the generesponsible for said naturally-occurring herbicide resistance can beidentified. The nucleic acid encoding the gene conferring thenaturally-occurring herbicide resistance can then be isolated. Theisolated nucleic acid comprises a gene or fragments thereof that encodesa protein responsible for causing the plant to be herbicide resistant.This isolated nucleic acid can then be used to (1) identify othernucleic acids which may contain naturally-occurring mutations thatprovide herbicide resistance to cotton plants; (2) introduce theisolated nucleic acid into a cotton plant which lacks herbicideresistance by means of genetic engineering which are known to theartisan of ordinary skill; (3) insert the isolated nucleic acid into asuitable vector which can be expressed in a cotton plant; and (4) insertthe vector into a plant cell (e.g., a cotton plant cell).

[0041] Vectors suitable for use in expressing the nucleic acids, whichwhen expressed in a plant confer herbicide resistance, include but arenot limited to pMON979, pMON977, pMON886, pCaMVCN, and vectors derivedfrom the tumor inducing (Ti) plasmid of Agrobaaerium tumefaciensdescribed by Rogers et al., Meth. Enzymol. 153:253-77 (1987). Thenucleic acid is inserted into the vector such that it is operably linkedto a suitable promoter. Suitable promoters for use with the nucleicacids include CaMV35S, ACTIN, NOS and PCSLV promoters.

[0042] The vectors comprising the nucleic acid can be inserted into aplant cell using a variety of known methods. For example, DNAtransformation of plant cells include but are not limited toAgrobacterium-mediated plant transformation, protoplast transformation,gene transfer into pollen, injection into reproductive organs, injectioninto immature embryos and particle bombardment. These methods aredescribed more fully in U.S. Pat. No. 5,756,290 and the references citedtherein. Site-specific recombination systems can also be employed toreduce the copy number and random integration of the nucleic acid intothe cotton plant genome. For example, the Cre/lox system can be used tomediate lox site-specific recombination in plant cells. This method canbe found at least in Choi et al., Nuc. Acids Res. 28: E19 (2000).

[0043] The resulting herbicide resistance is shown to be an infrequentlynaturally- occurring dominant genetic mutant and not the product ofgenetic modification. The process steps of the present invention havebeen found to enable the isolation of such genetic mutant in cotton on areliable basis. Such herbicide resistance is under genetic controlthrough the expression of one or more dominant gene pairs for herbicideresistance and can be readily transferred to other cotton varieties andlines, particularly when cotton plants are isolated and/or produced byconventional plant breeding.

[0044] The herbicide resistance of the present invention can be providedin true-breeding cotton varieties and lines as well as in F₁ cottonhybrids. When forming F₁ hybrids, the requisite genetic control isprovided in both parent plants (e.g., in cytoplasmic male sterile andrestorer parent plants). Also, cotton plants can be provided that areresistant to more than one herbicide when appropriatenaturally-occurring genes are incorporated into a single cotton plantsuch as by the use of conventional plant breeding followed by selection.

[0045] Heretofore, the use by cotton growers of herbicide resistance(e.g., glyphosate herbicide resistance) produced by genetic engineeringhas required the application of a herbicide only during the early stagesof cotton plant growth and not after the cotton plant has reached thefour-leaf stage. For instance, when herbicide-resistant cotton plants ofthis type are sprayed with a herbicide such as a glyphosate after thefour-leaf stage there is deleterious interference with cotton bollproduction. On the contrary, it has been found that herbicide resistantcotton plants of the present invention can be sprayed with herbicide atany stage of the plant life cycle without deleterious results. Forinstance, herbicide resistant cotton plants of the present invention canbe sprayed with a herbicide following squaring and flowering. A longerand safer period for spraying with a herbicide is provided. Accordingly,a cotton grower when utilizing cotton plants of the present invention,can spray the cotton field with herbicide whenever the need for weedcontrol is apparent without restriction with respect to timing. Thisprovides greater weed control options and flexibility to the cottongrower.

[0046] The following Examples are presented as specific illustrations ofthe claimed invention. It should be understood, however, that theinvention is not limited to the specific details of the Examples.

EXAMPLE I

[0047] Seeds of cotton B418 (Reg. No. PL-14, PI 583853) were selected asthe starting material. This variety was released jointly by the UnitedStates Department of Agriculture-ARS and the Texas AgriculturalExperiment Station and is publically available. See, Crop Science, Vol.35, No 5, Page 1518 (1995). The cotton seeds of this variety wereobtained from the United States Department of Agriculture at Weslaco,Tex., U.S.A., in 1994.

[0048] Approximately 100 grams of seeds of this variety were planted andthe agronomic characteristics and boll storm resistance were confirmedto be good. Such plants were allowed to undergo self-pollination and theresulting seeds were harvested.

[0049] Seeds of the variety were subjected to cold-stressing in a humidatmosphere for a period of seven days. More specifically, the seeds wereplaced in porous paper packets, and the packets were placed side by sidein a container that was present in a cold cabinet. The packetscontaining the seeds were contacted at the top and bottom by a dampcotton cloth, and a free end of the cotton cloth was immersed in acontainer of water. A drainage area was provided at the bottom of thecontainer and the seed packets were suspended above such area fordrainage. The temperature within the cold chamber was maintained at asubstantially constant 45° F. The relative humidity within the coldcabinet was approximately 99 percent. The seeds next were planted andgermination was attempted. Those seeds that germinated formed cottonplants that were self-pollinated to form more cotton seeds. The coldstressing was repeated on the harvested cotton seeds. These cotton seedswere next planted. The seeds that germinated were self-pollinated andcotton seeds were formed thereon. The resulting seeds were harvested andwere allowed to mature under dry room temperature conditions.

[0050] The mature cotton seeds next were soaked for a period of 6 hoursin a 2.5 percent by weight aqueous solution of glyphosate [i.e.,N-(phosphonomethyl)-glycine] that was provided at room temperature. Thisperiod of time was sufficient for the glyphosate herbicide to reach theembryos of the seeds as evidenced by the failure of many seeds togerminate and the early death of many seedlings resulting therefrom.

[0051] Approximately 90,000 cotton seeds next were planted in the fieldand germination was attempted. The plants that were formed were sprayedwith glyphosate herbicide at a rate of 2 pints per acre at the 4-leafstage and at the 10 to 12 leaf stage. Only 12 cotton plants survivedthis repeated subjection to the herbicide and displayed true herbicideresistance.

[0052] A portion of a young growing leaf from each plant was nextsubjected to a standard DNA-polymerase chain reaction analysis toconfirm the absence of a CaMV35S promoter that if present wouldattribute the manifest herbicide resistance to genetic engineering. Suchpromoter was found to be absent in a cotton plant that has beendesignated R418ctHG^(g)hg^(g). DNA sequence analysis also has confirmedthat this plant lacks the foreign Petunia gene present in commerciallyavailable herbicide resistant cotton varieties that are the product ofgenetic engineering. Genetic studies have shown that the manifestherbicide resistance is attributable to the dominant HG^(g) gene. Thisis a naturally-occurring mutant for herbicide resistance that has beendiscovered through the practice of the process of the present invention.Such gene when found in the heterozygous state can be provided in thehomozygous state (i.e., HG^(g) Hg^(g)) through conventional plantbreeding followed by selection as will be apparent to those skilled inplant breeding.

[0053] On Jun. 27, 2000 a deposit of 2,500 seeds of R418ctHG^(g)hg^(g)was made under the terms of the Budapest treaty at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, U.S.A., and has received ATCC Accession No. PTA-2132. Seedsfrom this deposit will be irrevocably made available upon the grant of apatent that makes reference to this deposit. However, the availabilityof these seeds is not to be construed as a license to practice theclaimed invention in contravention of rights granted under the authorityof any government in accordance with its patent or breeder's right laws.

EXAMPLE II

[0054] Example I is substantially repeated with the exception that seedsof cotton B-2-1ct are selected as the starting material. A more widelyplanted closely related variety derived from this source is JH216. Suchstarting material had undergone cold stressing prior to soaking in thesolution of glyphosate herbicide. Naturally-occurring glyphosateresistance is manifest in a resulting cotton plant designatedB-2-1ctHG^(g).

EXAMPLE III

[0055] Example II is substantially repeated with the exception that2,4-dichlorophenoxyacetic acid herbicide (i.e., 2,4-D) is substitutedfor the glyphosate herbicide. Naturally-occurring 2,4-D herbicideresistance is manifest in a resulting cotton plant designated B-2-1ctHG^(DID2) when sprayed at the 7 to 8-leaf stage with an aqueous 2.5percent by weight solution of 2,4-D herbicide at a rate of 2 pints peracre.

[0056] Cotton plants with 2,4-D herbicide resistance made possible bythe present invention will be advantageous to cotton growers where thisherbicide is being used to spray nearby corn fields in the event thereis unintended herbicide drift.

[0057] Although the invention has been described with reference topreferred embodiments, it is to be understood that variations andmodifications may be resorted to as will be apparent to those skilled inthe art. Such variations and modifications are to be considered withinthe purview and scope of the claims appended hereto.

I claim:
 1. A process for selecting a cotton plant which exhibitsgenetically-controlled herbicide resistance that is not attributable togenetic engineering comprising: (a) cold stressing cotton seeds in ahumid atmosphere, (b) planting said cotton seeds following step (a) toproduce cotton plants, (c) self-pollinating cotton plants produced instep (b) and forming cotton seeds thereon as the result of saidself-pollination, (d) maintaining said cotton seeds produced in step (c)for sufficient time to reach maturity, (e) soaking said mature cottonseeds from step (d) in a liquid comprising a herbicide for a period oftime sufficient for the herbicide to reach the embryos of the cottonseeds, (f) planting said cotton seeds following said soaking of step (e)in a growing medium and producing at least one cotton plant thatdisplays resistance to said herbicide, (g) analyzing a portion of aplant from at least one cotton plant produced in step (f) or adescendant plant thereof to confirm the absence of a foreign gene forherbicide resistance introduced by genetic engineering, and (h)selecting a cotton plant from step (g) which exhibitsgenetically-controlled herbicide resistance that is not attributable toa foreign gene for herbicide resistance introduced by geneticengineering.
 2. The process according to claim 1 wherein the duration ofsaid cold stressing of step (a) is carried out for at leastapproximately 7 days.
 3. The process according to claim 1 wherein saidcold stressing of step (a) is carried out at a temperature in the rangeof approximately 40 to 50° F.
 4. The process according to claim 1wherein said cold stressing temperature of step (a) is approximately 49°F. during the day and approximately 42° F. during the night and theduration of said cold stressing is at least approximately 7 days.
 5. Theprocess according to claim 1 wherein said herbicide of step (e) selectedfrom the group consisting of glyphosate, 2,4-dichlorophlenoxyaceticacid, glufosinate ammonium butanoic acid, and3,5-dibromo-4-hydroxybenzonitrile.
 6. The process according to claim 1wherein said herbicide of step (e) is glyphosate.
 7. The processaccording to claim 1 wherein said herbicide in step (e) is2,4-dichlorophenoxyacetic acid.
 8. The process according to claim 1wherein said herbicide in step (e) is glufosinate ammonium butanoicacid.
 9. The process according to claim 1 wherein said herbicide in step(e) is 3,5-dibromo-4-hydroxybenzonitrile.
 10. The process according toclaim 1 wherein during step (e) said liquid comprising a herbicide is anaqueous solution containing glyphosate in a concentration ofapproximately 2.5 percent by weight.
 11. The process according to claim1 wherein said soaking of step (e) has a duration of at least 6 hours.12. The process according to claim 1 wherein in step (f) said liquidcomprising a herbicide of step (e) is poured into the growing medium ofstep (f).
 13. The process according to claim 1 wherein the cotton plantproduced in step (f) is sprayed with said herbicide.
 14. The processaccording to claim 1 wherein the portion of the cotton plant that isanalyzed in step (g) is derived from a leaf.
 15. The process accordingto claim 6 wherein said analysis of step (g) confirms the absence ofCaMV35S promoter, ACTIN promoter, NOS promoter, and PCSLV promoter. 16.The process according to claim 6 wherein said analysis of step (g)confirms the absence of the Petunia gene for herbicide resistance. 17.The process according to claim 6 wherein said analysis of step (g)confirms the absence of the CP4 gene for herbicide resistance.
 18. Acotton seed capable of forming a cotton plant havinggenetically-controlled glyphosate herbicide resistance that isattributable to the homozygous gene pair HG^(g)HG^(g) obtainable fromcotton R418ctHG^(g)hg^(g) having ATCC Accession No. PTA-2132.
 19. Acotton plant having genetically-controlled glyphosate herbicideresistance that is attributable to the homozygous gene pair HG^(g)HG^(g)obtainable from cotton R418ctHG^(g)hg^(g) having ATCC Accession No.PTA-2132.
 20. A cotton plant which exhibits genetically-controlledherbicide resistance that is not attributable to a foreign gene forherbicide resistance introduced by genetic engineering obtained in step(h) of the process of claim 1 or descended therefrom in a subsequentgeneration.
 21. A cotton plant which exhibits genetically-controlledherbicide resistance that is not attributable to a foreign gene forherbicide resistance introduced by genetic engineering according toclaim 20, wherein glyphosate herbicide resistance is exhibited.
 22. Acotton seed which upon germination is capable of forming a cotton plantthat exhibits genetically-controlled herbicide resistance that is notattributable to a foreign gene for herbicide resistance introduced bygenetic engineering that is obtained from the selection made in step (h)of claim 1 or its descendants.
 23. A cotton seed which upon germinationis capable of forming a cotton plant that exhibitsgenetically-controlled herbicide resistance that is not attributable toa foreign gene for herbicide resistance introduced by geneticengineering according to claim 22 which is resistant to glyphosateherbicide.
 24. An isolated nucleic acid encoding for a protein whichwhen expressed in a cotton plant causes herbicide resistance that isnaturally-occurring in cotton wherein said isolated nucleic acid wasderived from the cotton plant of step (h) of claim 1 or its descendants.25. An isolated nucleic acid comprising a HG^(g) gene selected fromcotton R418ctHG^(g)hg^(g) having ATCC Accession No. PTA-2132 or itsdescendants and wherein said nucleic acid when expressed in a cottonplant causes said cotton plant to be glyphosate herbicide resistant. 26.A vector comprising the isolated nucleic acid of claim
 25. 27. A plantcell comprising the vector of claim 26.